It has been recognized that RNA viruses are useful in the genetic engineering of plants, particularly+-strand messenger sense RNA viruses. See, e.g., U.S. Pat. application Nos. 580,445 and the continuation-in-part application thereof filed simultaneously herewith, and 709,181, incorporated herein by reference.
A problem with prior methods involving the use of RNA viruses for genetic engineering of plants, however, has been the fact that many plant viruses providing suitable sites for the insertion of RNA copies of foreign genes are encapsidated in their naturally infective state in spherical or icosahedral capsids which impose geometric constraints on the amount of genetic material which can be carried by these viruses.
Examples of such useful viruses are the tripartite viruses of Tricornaviridae, such as brome mosaic virus (BMV), and cowpea chlorotic mottle virus (CCMV), which are packaged in icosahedral capsids. The BMV RNA3 segment has been extensively studied as a carrier for heterologous RNA; however, observation of the structure of functional elements of this RNA segment indicate that the 3.2-3.3 kb size of the natural BMV RNA1 is probably near the maximum size which can be carried by the icosahedral BMV coat.
BMV RNA3 is around 2.1 kb in size, so only around 1 kb at most of foreign sequence could be added to a packagable RNA3 derivative without deletion of a portion of the natural BMV RNA3 sequence. Although in the case of BMV RNA3, which has been extensively studied, identification of sequences which are not required in cis for replication and gene expression opens the possibility of constructing variants with such deletions, it is highly desirable for increased freedom and flexibility in making more sophisticated virus derivatives to be able to add heterologous RNA sequences without being required to delete an equal amount of the original viral RNA. The possibility also exists for BMV and some other isometric viruses that lower as well as upper RNA size limits exist for viral RNA packaging (Ahlquist et al. (1984) J. Mol. Biol. 172:369-383).
It would thus be desirable to encapsidate recombinant RNA viral sequences into a coat protein able to freely package pieces of RNA without upper or lower size constraints.
In contrast to the icosahedral viruses, a large class of plant viruses have elongated virions with the shape of rigid or flexuous rods. The tobacco mosaic virus is an example which has been extensively studied. See, e.g., M. H. V. Van Regenmortel (1981) "Tobamoviruses," in Handbook of Plant Virus Infections and Comparative Diagnosis, E. Kurstak (ed.), at 541-564; P. J. G. Butler (1984) "The Current Picture of the Structure and Assembly of Tobacco Mosaic Virus," J. Gen. Virol. 65:253-279; D. L. Beck, et al. (1985), "Synthesis of Full-length cDNA Clones of TMV," Phytopathology, 75:1334 (Abstract); and W. O. Dawson, et al. (1986) "cDNA Cloning of the Complete Genome of Tobacco Mosaic Virus and Production of Infectious Transcripts," Proc. Natl. Acad. Sci. U.S.A. 83:1832-1836.
The coat protein of such rod-shaped virions is encoded by an RNA gene. An assembly origin sequence is also necessary to initiate packaging. A cDNA clone containing the assembly origin of TMV Cowpea Strain (Cc) and coat protein gene has been isolated by T. Meshi, et al. (1981) "Nucleotide Sequence of a Cloned cDNA Copy of TMV (Cowpea Strain) RNA, Including the Assembly Origin, the Coat Protein Cistron, and the 3' Non-Coding Region," Mol. Gen. Genet. 184:20-25. In this particular TMV strain, the assembly origin sequences are within the coat protein gene; however, in other strains, the assembly origin and coat protein genes are separate.
The coat proteins of rod-shaped virions are assembled in a helical array, and encapsidate the RNA rod-shaped viral particle with RNA wound helically within the interior of the extendable particle. Thus, in order to increase the size range of genetically engineered RNA viral sequences, it is an object of this invention to provide a recombinant RNA sequence encapsidated in a rod-shaped coat.
In general, it would be desirable to package viral sequences in any heterologous coat protein capsid, for example when the desired host has established immunities against the natural coat protein of the viral sequence with which infection is desired. However, prior to this invention, no hybrid viruses combining functions (e.g., host specificity, infectivity, and the like) derived from more than one RNA virus had been constructed.
A viable recombinant was constructed at the cDNA level between poliovirus types 1 and 3 which are 70% homologous in RNA sequence (G. Stanway et al. (1986), "Construction of poliovirus intertypic recombinants by use of cDNA," J. Virol. 57:1187. This recombinant carried the 0.74 kb 5' untranslated sequence and first 11 polyprotein codons from type 3, with the remainder of the sequence from type 1. A second recombinant reported involved insertion of a portion of the VP1 capsid protein gene from type 3 into a type 1 context, but this was non-viable, indicating problems in combining non-homologous functional regions.
An additional example of recombination between divergent Picornavirus genomes was the construction of a recombination between poliovirus type 1 and coxsackie B3 virus (B. L. Semler et al. (1986), "A chimeric plasmid from cDNA clones of poliovirus and coxsackie virus that is temperature-sensitive," Proc. Natl. Acad. Sci. USA 83:1777). In this case a 0.4 kb segment of the coxsackie B3 5' noncoding region was inserted in a poliovirus context, replacing a segment with which it had 70% homology. The resulting recombinant virus was viable but showed a temperature-sensitive replication phenotype.
In none of these reported attempts to produce recombinant viruses was a viable hybrid virus produced in which a substitute segment has less than 70% homology with the segment it replaced, and in no case were separate functions from two or more different viruses successfully recombined. N or has the artificial construction of a hybrid RNA plant virus from two distinct viral types been reported.
Unencapsidated derivatives of rod-shaped viruses have been recently used as vectors to carry a foreign gene in recombinant RNA (Takamatsu, N. et al., "TMV-RNA mediated foreign gene expression in tobacco plants," In press), and recombinant RNA sequences have been encapsidated in a rod-shaped coat in vitro (Sleat, D. E. et al., "Packaging of Recombinant RNA Molecules into Pseudovirus Particles Directed by the Origin-of-Assembly Sequence from Tobacco Mosaic Virus RNA," In press). However, the in vivo packaging of a recombinant viral RNA in a capsid foreign to the infective viral sequences has not been previously reported.